The role and medical prospects of long non-coding RNAs in cardiovascular disease.

Cardiovascular disease (CVD) has reached epidemic proportions and is a leading cause of death worldwide. One of the long-standing goals of scientists is to repair heart tissue damaged by various forms of CVD such as cardiac hypertrophy, dilated cardiomyopathy, myocardial infarction, heart fibrosis, and genetic and developmental heart defects such as heart valve deformities. Damaged or defective heart tissue has limited regenerative capacity and results in a loss of functioning myocardium. Advances in transcriptomic profiling technology have revealed that long noncoding RNA (lncRNA) is transcribed from what was once considered "junk DNA." It has since been discovered that lncRNAs play a critical role in the pathogenesis of various CVDs and in myocardial regeneration. This review will explore how lncRNAs impact various forms of CVD as well as those involved in cardiomyocyte regeneration. Further, we discuss the potential of lncRNAs as a therapeutic modality for treating CVD.

Spatiotemporal expression of long noncoding RNA Moshe modulates heart cell lineage commitment.

The roles of long non-coding RNA (LncRNA) have been highlighted in various development processes including congenital heart defects (CHD). Here, we characterized the molecular function of LncRNA, Moshe (1010001N08ik-203), one of the Gata6 antisense transcripts located upstream of Gata6, which is involved in both heart development and the most common type of congenital heart defect, atrial septal defect (ASD). During mouse embryonic development, Moshe was first detected during the cardiac mesoderm stage (E8.5 to E9.5) where Gata6 is expressed and continues to increase at the atrioventricular septum (E12.5), which is involved in ASD. Functionally, the knock-down of Moshe during cardiogenesis caused significant repression of Nkx2.5 in cardiac progenitor stages and resulted in the increase in major SHF lineage genes, such as cardiac transcriptional factors (Isl1, Hand2, Tbx2), endothelial-specific genes (Cd31, Flk1, Tie1, vWF), a smooth muscle actin (a-Sma) and sinoatrial node-specific genes (Shox2, Tbx18). Chromatin Isolation by RNA Purification showed Moshe activates Nkx2.5 gene expression via direct binding to its promoter region. Of note, Moshe was conserved across species, including human, pig and mouse. Altogether, this study suggests that Moshe is a heart-enriched lncRNA that controls a sophisticated network of cardiogenesis by repressing genes in SHF via Nkx2.5 during cardiac development and may play an important role in ASD.

Combination of small RNAs for skeletal muscle regeneration.

Selectively controlling the expression of the target genes through RNA interference (RNAi) has significant therapeutic potential for injuries or diseases of tissues. We used this strategy to accelerate and enhance skeletal muscle regeneration for the treatment of muscular atrophy. In this study, we used myostatin small interfering (si)RNA (siGDF-8), a major inhibitory factor in the development and postnatal regeneration of skeletal muscle and muscle-specific microRNAs (miR-1 and -206) to further accelerate muscle regeneration. This combination of 3 small RNAs significantly improved the gene expression of myogenic regulatory factors in vitro, suggesting myogenic activation. Moreover, cell proliferation and myotube formation improved without compromising each other, which indicates the myogenic potential of this combination of small RNAs. The recovery of chemically injured tibialis anterior muscles in rats was significantly accelerated, both functionally and structurally. This novel combination of siRNA and miRNAs has promising therapeutic potential to improve in situ skeletal muscle regeneration.

Transcription Factors Oct-1 and GATA-3 Cooperatively Regulate Th2 Cytokine Gene Expression via the RHS5 within the Th2 Locus Control Region.

The T helper type 2 (Th2) locus control region (LCR) regulates Th2 cell differentiation. Several transcription factors bind to the LCR to modulate the expression of Th2 cytokine genes, but the molecular mechanisms behind Th2 cytokine gene regulation are incompletely understood. Here, we used database analysis and an oligonucleotide competition/electrophoretic mobility shift assays to search for transcription factors binding to RHS5, a DNase I hypersensitive site (DHS) within the Th2 LCR. Consequently, we demonstrated that GATA-binding protein-3 (GATA-3), E26 transformation-specific protein 1 (Ets-1), octamer transcription factor-1 (Oct-1), and Oct-2 selectively associate with RHS5. Furthermore, chromatin immunoprecipitation and luciferase reporter assays showed that Oct-1 and Oct-2 bound within the Il4 promoter region and the Th2 LCR, and that Oct-1 and GATA-3 or Oct-2 synergistically triggered the transactivational activity of the Il4 promoter through RHS5. These results suggest that Oct-1 and GATA-3/Oct-2 direct Th2 cytokine gene expression in a cooperative manner.

Effects of glucocorticoid receptor small interfering RNA delivered using poly lactic-co-glycolic acid microparticles on proliferation and differentiation capabilities of human mesenchymal stromal cells.

Bone marrow-derived mesenchymal stem cells (MSC) are a potential attractive source of cells for stem cell-based tissue regeneration, but the small number and reduced capabilities of MSC proliferation and differentiation due to in vitro replicative senescence and donor-associated pathophysiological factors, including age and estrogen depletion, severely restrict their potential usefulness in clinical applications. Glucocorticoids (GC) are well-known steroid hormones that regulate MSC proliferation and differentiation, but the defined effects and underlying mechanisms of endogenous glucocorticoids on MSC characteristics are not understood. This study investigated the effects of the blockage of endogenous GC using glucocorticoid receptor (GR) small interfering RNA (siRNA) delivered using biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles on proliferation and differentiation capabilities of human MSC in vitro. The results show that we can prepare PLGA microparticles as a delivery system for GR siRNA and maintain release of siRNA up to 40 days in vitro. Transfection of GR siRNA significantly downregulates GR and upregulates the expression of fibroblast growth factor-2 and Sox-11 of human MSC. MSC that have proliferated with endogenous GC blocked in vitro have greater proliferation rates and exhibit upregulated expression of osteogenic markers (alkaline phosphatase and core binding factor alpha 1) under differentiation stimulation after 1 week. Under adipogenic differentiation, MSC proliferated in vitro with siRNA transfection, resulting in significantly lower adipogenic markers (peroxisome proliferator-activated receptor and lipoprotein lipase) than controls. In conclusion, PLGA particles can serve as a tool for delivery of GR siRNA to effectively block the effects of endogenous GC on MSC, which has the potential to improve the capabilities of human MSC for clinical application by preventing replicative senescence.

Synthesis and characterization of mannosylated pegylated polyethylenimine as a carrier for siRNA.

Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for research and treatment of numerous diseases. In this study, we develop and characterize a delivery system for siRNA composed of polyethylenimine (PEI), polyethylene glycol (PEG), and mannose (Man). Cationic PEI complexes and compacts siRNA, PEG forms a hydrophilic layer outside of the polyplex for steric stabilization, and mannose serves as a cell binding ligand for macrophages. The PEI-PEG-mannose delivery system was constructed in two different ways. In the first approach, mannose and PEG chains are directly conjugated to the PEI backbone. In the second approach, mannose is conjugated to one end of the PEG chain and the other end of the PEG chain is conjugated to the PEI backbone. The PEI-PEG-mannose delivery systems were synthesized with 3.45-13.3 PEG chains and 4.7-3.0 mannose molecules per PEI. The PEI-PEG-Man-siRNA polyplexes displayed a coarse surface in Scanning Electron Microscopy (SEM) images. Polyplex sizes were found to range from 169 to 357 nm. Gel retardation assays showed that the PEI-PEG-mannose polymers are able to efficiently complex with siRNA at low N/P ratios. Confocal microscope images showed that the PEI-PEG-Man-siRNA polyplexes could enter cells and localized in the lysosomes at 2h post-incubation. Pegylation of the PEI reduced toxicity without any adverse reduction in knockdown efficiency relative to PEI alone. Mannosylation of the PEI-PEG could be carried out without any significant reduction in knockdown efficiency relative to PEI alone. Conjugating mannose to PEI via the PEG spacer generated superior toxicity and gene knockdown activity relative to conjugating mannose and PEG directly onto the PEI backbone.

Inhibition of histone deacetylation enhances the neurotoxicity induced by the C-terminal fragments of amyloid precursor protein.

The AICD (APP intracellular Domain) and C31, caspase-cleaved C-terminal fragment of APP, have been found in Alzheimer's disease (AD) patients' brains and have been reported to induce apoptosis in neuronal cells. In recent, the C-terminal fragments of amyloid precursor protein (APP-CTs) have been reported to form a complex with Fe65 and the histone acetyltransferase Tip60 and are thought to be involved in gene transcription. In this study, based on the hypothesis that APP-CTs might exert neurotoxicity by inducing some gene transcription, we investigated the effects of APP-CTs on histone acetylation which indicates that transcription is actively going on and also on the relationship between histone acetylation and the cytotoxicity induced by APP-CTs in nerve growth factor (NGF)-differentiated PC12 cells and rat primary cortical neurons. Here we demonstrate that the expression of APP-CTs [C31, AICD (C59) and C99] induces increases in acetylation of histone 3 and histone 4 and that treatment with sodium butyrate, an inhibitor of histone deacetylase, significantly enhances the cytotoxicity induced by APP-CTs. The acetylation of histone plays an important role in allowing regulatory proteins to access DNA and is likely to be a major factor in the regulation of gene transcription. Taken together, our results suggest that APP-CTs exert neurotoxicity by transcription-dependent mechanisms and this might contribute to the pathogenesis of AD.